The tick borne flaviviruses (TBFV) includes Tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Powassan virus and Langat virus (LGTV). The TBFV are listed among the NIAID category B and C lists of priority for research on pathogenesis, to identify novel targets for therapeutic and vaccine development. As their name suggests, these viruses are transmitted by ticks, and following infection of humans, cause encephalitis, meningitis or hemorrhagic fevers resulting in approximately 13,000 hospitalizations annually with mortality rates as high as 40%. [unreadable] [unreadable] The TBFV belong to the Family Flaviviridae, genus Flavivirus, which comprise some of the most medically significant emerging and re-emerging pathogens. Other members include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses.[unreadable] [unreadable] The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the tick and the mammal) critical to virus replication and pathogenesis. We have been studying LGTV which is a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. This makes LGTV an excellent model to gain insight into the TBFV. In particular, our laboratory has a number of ongoing projects outlined below to study interactions between the TBFV and their hosts, with an emphasis on host innate immunity.[unreadable] [unreadable] 1. Interactions between TBFV and host innate responses to infection.[unreadable] Following the bite from an infected tick, dendritic cells (DCs) resident in the skin of the mammalian host are among the first cell types infected by TBFV. These cells have important roles in innate immunity through the production of interferon (IFN), cytokines and chemokines, as well as in orchestrating adaptive immunity. Thus, early interactions between these cells and TBFV are likely to have a major influence on the outcome of infection. We have initiated a project in the lab to investigate the effects of TBFV infection on primary DCs and the ability of TBFV to modulate the signal transduction pathways involved in detecting virus infection and producing IFN. [unreadable] [unreadable] Following infection with LGTV, flow cytometry was used to distinguish between infected and uninfected bystander DCs. Maturation profiles revealed that infected DC, but not bystander cells, increased cell surface expression of MHC class II. However, infected DC did not upregulate CD80 and CD86, costimulatory molecules involved in activating T cell responses. Moreover, infected DC did not respond appropriately to stimulation with toll-like receptor (TLR) 3 or TLR4 ligands. The failure of LGTV-infected DC to express key molecules involved in anti-viral immunity in vivo may contribute to viral evasion of immune responses. Hence, examining these interactions will lead to an understanding of the mechanisms that contribute to TBFV pathophysiology.[unreadable] [unreadable] Type I (IFN and IFN) and type II (IFN) IFNs are crucial elements of the innate immune response to flavivirus infection, restricting virus replication, dissemination and lethality in mouse models. Type I IFN treatment of humans is a leading therapeutic candidate for flavivirus infection. However, such treatment often fails. We have shown that LGTV interferes with IFN signaling by directly inhibiting Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction. In this manner, LGTV is similar to the mosquito-borne flaviviruses WNV, JEV, DEN and YFV, which can also inhibit IFN-mediated JAK-STAT signaling. Our laboratory demonstrated that the nonstructural protein, NS5, of LGTV suppresses JAK-STAT signaling in response to both type I and type II IFN. We have investigated the relative IFN antagonist ability of NS5 and another nonstructural protein NS4B (implicated in IFN antagonism for DEN) from different flaviviruses including WNV and JEV. These studies have identified NS5 as a major IFN antagonist for virulent strains of WNV in addition to the already known role of NS5 in IFN-evasion by JEV. NS5 from attenuated strains of both WNV and JEV had a lower intrinsic ability to suppress signaling than NS5 from virulent strains, suggesting that NS5 may be a flavivirus virulence determinant. Furthermore, we have mapped specific residues within LGTV NS5 responsible for IFN antagonism. Taken together, this work will provide further insight into the importance of IFN-evasion in flavivirus pathogenesis as well as contribute to the next generation of live-attenuated vaccine design.[unreadable] [unreadable] 2. Study of virus interactions with the invertebrate host. [unreadable] In addition to working with mouse models of infection, we have developed a novel method of infecting tick larvae with LGTV by immersion. This extremely versatile method permits synchronous infection of large numbers of ticks with a defined virus inoculum, and without a requirement for that virus to establish an infection and viremia in mice. This method not only enables study of virus replication within the initial infected tick larvae and nymphs, but also results in efficient trans-stadial transmission as well as horizontal transmission to C57BL/6 mice. Hence, the immersion method of infection is a powerful tool to study viral and host determinants for pathogenesis in both ticks and in the mammalian host. We are currently utilizing the immersion method of infection to examine global transcriptional responses of the tick, Ixodes scapularis, to infection with LGTV using Agilent microarrays. The aim of this work is to identify tick host proteins important for the replication or transmission of TBFV. These proteins can be used for the development of novel anti-tick vaccines that prevent virus transmission to the immunized mammalian host. [unreadable] [unreadable] We are also interested in identifying virus determinants that confer a selective replication advantage in either tick or mammalian systems and understanding how these determinants influence pathogenesis in the mammalian host. To examine these questions, we derived two LGTV variants by repeatedly passaging the virus in tick or mammalian cell culture, followed by sequencing of the virus genome. To summarize this work, we identified two clusters of coding changes in the LGTV genome associated with host adaptation and altered pathogenesis in a mouse model of infection. The first was in the envelope (E) protein while the second was in the region encompassing NS3, NS4A and NS4B. We plan to use the two animal models of infection, both tick and mouse, in combination with reverse genetics to determine the relative contribution of these genetic changes to virus transmission and virulence.